The present invention relates to detection of a nucleic acid by hybridization. In particular, the present invention relates to a method for detecting a nucleic acid by hybridization as well as a nucleic acid and a nucleic acid-immobilized substrate used for the method.
In the fields of clinical test, food inspection, medicolegal test and so forth, as methods for detecting and identifying biologically active substances such as nucleic acids, antibodies and antigens present in specimens, nucleic acid probe methods, enzyme immunoassays and so forth are used depending on the target substance.
As methods utilizing detection of nucleic acids, there can be mentioned identification of microbial strains of pathogenic microbes etc., DNA identification in the medical jurisprudence and so forth. In these method, detection is usually carried out as follows. A nucleic acid having a sequence complementary to a target nucleic acid is labeled directly with an enzyme or the like, or labeled indirectly by using a hapten or the like. This labeled nucleic acid is hybridized with a target nucleic acid. After the labeled nucleic acid that does not hybridize is removed or its labeled portion is inactivated, the labeled portion of the hybridized target nucleic acid is detected to confirm presence and amount of the target nucleic acid.
In the conventional nucleic acid detection methods, it is extremely important to immobilize nucleic acids on a surface of solid phase such as tubes, microtiter plates, membrane filters and beads. Therefore, various methods have been published for immobilization of nucleic acids.
There are known, for example:    (1) a method of chemically bonding a nucleic acid into which a modification group is introduced, to a substrate, such as immobilization by a disulfide bond between a nucleic acid having a thiol group at its 5′ end and a bead-like substrate having thiol groups (P. J. R. Day, P. S. Flora, J. E. Fox, M. R. Walker, Biochem. J., 278, 735–740 (1991));    (2) a method of attaining immobilization by physical adsorption, such as immobilization of nucleic acid by adsorption on nitrocellulose, poly-L-Lysine, nylon membrane or the like through UV irradiation or heat treatment (J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning, Cold Spring Harbor Laboratory Press, Second Edition, pages 2.109–2.113 and pages 9.34–9.46, International Patent Publication in Japanese (Kohyo) No. 10-503841), and immobilization by physical adsorption on a microplate (G. C. N. Parry and A. D. B. Malcolm, Biochem. Soc. Trans., 17, 230–231 (1989));    (3) a method of synthesizing DNA on a substrate using a nucleotide bonded to a substrate (WO97/10365), and so forth.
However, these methods have drawbacks, i.e., in the method of (1), extremely special apparatuses and reagents are required, and in the method of (2), nucleic acids are dropped off from the substrate during the hybridization procedure, in particular, in operation processes, and thus detection sensitivity may be reduced or reproducibility may not be obtained. Furthermore, this method has another drawback, that is, although a long nucleic acid can be immobilized, a short nucleic acid of about 50-mer or less such as oligomers cannot be efficiently immobilized.
Further, in the method of (3), extremely special apparatuses and reagents are required for synthesizing DNA on a substrate, and the nucleic acid synthesized by this method is limited to about 25-mer or less.